The long-term goal of this project is to understand the mechanisms that are involved in the increased incidence of cardiovascular complications in Type 2 diabetes. Vascular dysfunction manifested as enhanced vasoconstriction and/or impaired vasorelaxation occurs in diabetes. Endothelial factors such as increased endothelin-1 (ET-1) as well as decreased nitric oxide (NO) bioavailability as a result of excess formation of superoxide (O2.-) in diabetes contributes to vascular dysfunction. ET-1 exerts potent vasoconstrictor actions and stimulates 02.- formation via activation of ETA receptors, which are balanced by endothelial ETB receptors that mediate opposite effects. However, the functional relationship between ETA/ETB ratio and vascular function in diabetes has yet to be fully explored. We have evidence that vasodilatation in ET-1-precontracted arteries is markedly impaired and that endothelial ETB receptor density is lower in diabetic patients. Our pilot data shows that ET-1 causes enhanced O2.- production in this patient population. Accordingly, the first hypothesis is that endothelial ETB receptors counterbalance ETA-mediated excess O2.- generation in diabetes. To test this hypothesis, we will a) determine O2.- production in the mesenteric beds of a spontaneously diabetic (Type 2) Goto-Kakizaki rat model treated with an ETA or ETB antagonist; and b) determine O2.- production in a human endothelial/vascular smooth muscle co-culture model in which varying degrees of endothelial ETB blockade is established by a ETB receptor antagonist. We predict that ETA blockade will prevent O2.- generation in diabetes whereas ETB blockade will worsen O2.- production. To test the hypothesis that excess ETA-mediated O2.- production will impair vasodilatation in diabetes, we will a) determine the vasodilating capacity of mesenteric beds of Goto-Kakizaki rat model treated with an ETA or ETB antagonist and investigate whether scavenging O2.- normalizes the vasodilating capacity in untreated diabetic rats. We predict that ETA blockade or scavenging of O2.- will restore and ETB blockade will worsen the vasorelaxation capacity in diabetes. The current research proposal was developed in response to Program Announcement PA-03-053 entitled "Academic Research Enhancement Award," The primary goal of this PA is to support the development of health-related meritorious research projects including pilot studies while fostering a research environment for students. Therefore, we have taken a translational research approach, incorporating our important findings with human tissue into animal and cell culture models to specifically test our hypotheses while providing training and career development for both Pharm.D. and Ph.D. candidates to clinically oriented research. As an outcome of the proposed translational research project we expect to have generated new and important information related to the roles of ETA and ETB receptors on vascular dysfunction underlying diabetic complications and have identified a potentially new therapeutic target, which would provide a solid framework to build upon more mechanistic studies as well as designing a vascular protection strategy in diabetes. The results will lead to the development of novel therapeutic targets for the high-risk diabetic patients and may identify subgroups of patients that would be more suitable for these new treatment strategies.